Many diagnostic tests used in the current art involve the amplification of a target nucleic acid. Respective amplification based assays are in particular used in order to detect the presence or absence of a target nucleic acid, such as for example a pathogen nucleic acid, in a biological sample material. If the target nucleic acid is comprised in the sample, a signal is obtained in the amplification reaction thereby indicating that the target nucleic acid is present and that, for example, a patient is infected with a certain pathogen. There is a high demand to control that the assay was performed accurately and accordingly, and thus provides a reliable result.
A major problem in particular in the field of clinical diagnostics is the occurrence of false negative results. False negatives may occur for several reasons. E.g. false negatives may occur due to a failure of one or more reagents that are used in the amplification reaction, due to a failure of thermal cycling or by an inhibition of the amplification reaction. However, false negatives are also attributable to that no or not enough sample material was subjected to the assay. The transfer of sample material is a critical step, in particular in high-throughput applications and when performing the respective tests using automated platforms. E.g. the biological sample such as a swab sample is transferred into the lysis composition by wiping off, shaking or similar means. Such transfers are prone to errors because no or not enough sample material may be transferred in order to allow a valid interpretation of the test results. Similar problems can also occur when handling liquid biological samples, e.g. when pipetting errors occur. The occurrence of false negatives is a major problem in the field of clinical diagnostics, e.g. because necessary treatments cannot be initiated in due time or subjects infected with a certain pathogen are not properly identified. E.g. in hospitals incoming patients are often routinely screened for the presence of certain pathogens such as MRSA and in case of a positive result are put into quarantine. A false negative in such a setting would allow the spread of the disease/pathogen.
In order to identify false negative assay results that are due to failure of the amplification reaction, it is common in the prior art to include an internal control in each amplification reaction. Thus, for each analysed biological sample, a respective internal control is usually included in the amplification reaction. As internal control, nucleic acids such as DNA or RNA (in case of a reverse transcription amplification) are commonly used. A respective internal control nucleic acid is either added separately to the amplification reaction or it can be directly included in the reagents that are used for performing the amplification. For example, a respective internal control can be comprised in the amplification master mix. Primers and/or probes for detecting the respective internal control are also added. Likewise, they can be added separately to the amplification reaction or may be comprised in the amplification master mix. If the amplification reaction works properly, an amplification signal is obtained, thereby indicating that the amplification reaction is valid. Thereby, false negatives that are due to a failure of the amplification reaction can be properly identified because if the amplification reaction did not work accurately, no amplification signal is obtained for the internal control.
However, a respective internal control cannot identify false negatives that occur because either no or not enough sample material was subjected to the assay, because also in this case an amplification signal will be obtained for the internal control because the control is added separately from the biological sample to the amplification reaction. However, the lack of sufficient sample material in the amplification assay can be an important error source that leads to false negative results. In particular, automated protocols that are performed using robotic systems are susceptible to such errors, for example because the sample transfer, e.g. the pipetting, did not work accurately what may result in that either no or not enough sample material was subjected to the assay in order to provide a reliable result. Such problems also occur if the samples are processed manually, in particular in high-throughput applications. Furthermore, for many sample types such as for example swab samples, the transfer of the sample material is particularly difficult. If no sufficient sample material is subjected to the lysis reaction and thus to the amplification reaction, this leads to false negative results in the assay.
Therefore, there is a high demand for methods that allow to identify false negatives and thus invalid assays that are due to that no sufficient sample material was subjected to the amplification based analytical assay.
It is the object to overcome at least one drawback of the prior art methods. In particular, it is the object of the present invention to provide a simple method for controlling that a biological sample material was included in an amplification-based assay. Furthermore, it is the object of the present invention to provide an improved amplification assay, which allows to identify false negatives that occur because insufficient sample material was subjected to the assay.